This invention relates to a method of making phenyl acetyl carbinol (PAC), which is useful as an intermediate in the manufacture of l-ephedrine and d-pseudoephedrine. This invention also relates to microorganisms especially adapted for use in the manufacture of phenyl acetyl carbinol and to a method for providing the microorganisms.
Pseudoephedrine and ephedrine are two major medicinal chemicals. Pseudoephedrine is useful as a nasal decongestant and is found as an ingredient in cough and cold capsules, sinus medications, nose sprays, nose drops and allergy and hay fever medications. Ephedrine is useful as a topical nasal decongestant, a treatment for mild forms of shock (CNS stimulant) and as a bronchodilator.
L-ephedrine is a natural product found in various species of plants. L-ephedrine is obtained from dried plant material by an initial treatment with alkali followed by extraction with organic solvent. While d-pseudoephedrine is also found in nature, it is more easily obtained in high yield from l-ephedrine by Welsh rearrangement.
L-phenyl acetyl carbinol (PAC) is the key intermediate in the synthesis of l-ephedrine. The transformation of benzaldehyde to L-(-)phenyl acetyl carbinol by Brewer's yeast was first described by Newberg and Hirsch. Biochem. Z., 115: 282-310 (1921). More particularly, benzaldehyde can be transformed by a fermenting yeast into L-(-)phenyl acetyl carbinol as follows: ##STR1##
The combination of yeast transformation of benzaldehyde to produce PAC and chemical conversion of the PAC to make l-ephedrine is described in U.S. Pat. No. 1,956,950. The PAC can be converted by a chemical reductive amination with methylamine to optically pure L-ephedrine as follows: ##STR2## The l-ephedrine can then converted in high yield to d-pseudoephedrine as follows: ##STR3## It is apparent from this reaction scheme that microbial transformation of benzaldehyde by yeast to form L-(-)phenyl acetyl carbinol in high yield and purity is of prime importance for successful commercial operation of the synthetic route.
Prior processes for the production of PAC from yeast involve the addition of the yeast to a medium containing molasses, beer wort, MgSO.sub.4 and other salts at a pH of 5.5-6.0. After an initial short period of stirring and aeration, a mixture of acetaldehyde and benzaldehyde is added in portions. A final concentration of PAC of about 7.5 g/L is obtained in 5-10 hours of continued stirring and aeration. With brewer's or baker's yeast, benzyl alcohol is always observed as a co-product. The highest reported yield of PAC based on benzaldehyde is about 73%. The remaining benzaldehyde is converted to the alcohol. Acetaldehyde is not essential for PAC production, but addition of this compound is required in order to achieve the highest yields of PAC.
Most of the literature concerning the synthesis of PAC by fermenting yeast deals with yield optimization. There is a general concensus that high levels of yeast are needed to obtain relatively low levels of PAC. The available literature suggests that the current yeast transformation of benzaldehyde to PAC is inefficient and yeast productivity is low. The yeast cannot be used for multiple batches because PAC production drops with increased exposure to the substrates and to the end product.
In addition, current yeast transformation provides only low concentrations of PAC in the fermentation liquor. This requires large process volumes and consequently large volumes of extraction solvent, which adversely impact on labor and utility costs in commercial operations.
In addition, the yield of PAC from benzaldehyde is decreased as a result of the catalytic reduction of benzaldehyde by an alcohol dehydrogenase to form benzyl alcohol, which is an unwanted by-product. All of the PAC-producing strains that have been examined produce benzyl alcohol.
Accordingly, there exists a need in the art for an improved method of making PAC by yeast transformation of benzaldehyde. The method should provide a higher yeast productivity and higher maximum concentrations of